In the Japanese official detection method for unauthorized genetically modified (GM) papayas, one of two types of real-time PCR reagents with DNA polymerase (TaqMan Gene Master Mix [TaqMan Gene] or FastGene QPCR Probe Mastermix w/ROX [FastGene]) is primarily used for measurement. In 2022, we conducted a laboratory performance study on the unauthorized GM papaya line PRSV-YK, and the results revealed that high threshold cycle (Cq) values for the PRSV-YK detection test were obtained using TaqMan Gene with the 7500 Fast & 7500 Real-Time PCR System (ABI7500) and QuantStudio 12K Flex (QS12K), indicating the possibility of false negatives. The possibility of similar problems with all unauthorized GM papaya lines detection tests needs to be evaluated. In this study, we performed detection tests on unauthorized GM papaya lines (PRSV-YK, PRSV-SC, and PRSV-HN), the cauliflower mosaic virus 35S promotor (CaM), and a papaya positive control (Chy), and examined how the limits of detection (LOD) for each test are affected by two types of DNA polymerases (TaqMan Gene and FastGene) and three types of real-time PCR instruments (ABI7500, QS12K, and LightCycler 480 Instrument II [LC480]). In the PRSV-YK and PRSV-SC detection tests using ABI7500 and QS12K, measurement with TaqMan Gene showed a higher LOD than FastGene. In this case, an exponential amplification curve was confirmed on the amplification plot; however, the amplification curve did not cross the ΔRn threshold line and the correct Cq value was not obtained with a threshold line=0.2. The other tests (PRSV-HN, CaM, and Chy with ABI7500 and QS12K, and all detection tests with LC480) showed no important differences in the LOD for each test using either DNA polymerase. Therefore, when performing PRSV-YK and PRSV-SC detection tests with the ABI7500 or QS12K, FastGene should be used to avoid false negatives for foods containing GM papaya lines PRSV-YK and PRSV-SC at low mixing levels.

Since the establishment of procedures for the safety assessment of food products that use recombinant DNA technology, the manufacture, import, and sale of genetically modified (GM) foods that have not undergone safety assessment are prohibited under the Food Sanitation Act. Therefore, a performance study to confirm the GM food testing operations of each laboratory is very important to ensure the reliability of the GM food monitoring system. In 2022, GM papaya line PRSV-YK-which has not yet been authorized in Japan-was selected for testing, and a papaya paste and a DNA solution were used as the test samples. With these samples, a laboratory performance study of the DNA extraction and real-time PCR operations was conducted. This confirmed that the 18 participating laboratories were generally performing the DNA extraction and real-time PCR operations correctly. However, some laboratories using certain DNA amplification reagent with some real-time PCR instruments were not able to determine the PRSV-YK detection test. This suggests that the PRSV-YK detection test may not be able to correctly detect samples containing GM papaya when performed with these combinations of instruments and reagent. In order to ensure the reliability of the PRSV-YK detection test, it is necessary to examine in detail how the combination of DNA polymerase reagents and real-time PCR instruments affects the detection limit, and to implement an appropriate solution.

Periodontal diseases, as an important part of oral pathology, present different characteristics when affecting children and adolescents or young adults. Studies have shown that adolescence and childhood are closely related to a high risk of periodontal disease, but the follow-up for periodontal health or damage at this age has been insufficiently appreciated until now. The aim of this study was to identify subgingival microorganisms using a real-time polymerase chain reaction (PCR) in a group of children and adolescents aged 7-17 years with and without cardiovascular disease. The group of 62 subjects with gingival inflammation and poor hygiene was divided into two groups according to general condition: 31 subjects with carduivascular disease (group A) and 31 subjects without cardiovascular disease (group C). Subjects were examined in the initial consultation, the state of hygiene and periodontal inflammation was assessed using the plaque index (PI) and gingival index (GI), and samples were taken from the gingival sulcus using sterile paper cones to determine nine subgingival microorganisms. Nine subgingival microorganisms were identified: Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Treponema denticola (Td), Tannerella forsythias (Tf), Prevotella intermedia (Pi), Peptostreptococcus (Micromonas) micros (Pm), Fusobacterium nucleatum (Fn), Eubacterium nodatum (En), and Capnocytophaga gingivalis (Cg). The patients were included in a specialist treatment program which aimed to relieve the inflammatory condition, remove local irritative factors, and train the patients to perform proper oral hygiene at home by using primary and secondary oral hygiene products. Subjects were reevaluated 3 months after treatment, when measurements for the PI and GI and microbiological determinations were repeated. The results showed a predominance of subjects aged 16-17 years (12.4%). Among the subjects with marked gingival inflammation, the male gender was predominant (58.06%). The PI values changed considerably after treatment, with lower values in patients presenting a general condition without cardiovascular disease (PI = 8.10%) compared with the patients with cardiovascular disease (PI = 13.77%). After treatment, the GI showed considerable changes in both groups. Red, orange, and purple complex microorganisms were found before treatment and decreased considerably after treatment in both groups. The highest values were found for Treponema denticola (140,000 (1.4 × 105)) in patients with cardiovascular disease and generalized gingival inflammation. Of the pathogenic microorganisms, the most common was Tannerella forsythia in 52 patients before treatment, and red microorganisms considerably appeared in only 10 patients after treatment. Capnocytophaga gingivalis remained constant both in the diseased state and after treatment and was consistent with periodontal health. Children with cardiovascular diseases had a higher prevalence of gingival manifestations. The composition of the subgingival microbial plaque was directly influenced by the degree of oral hygiene, but the response to specialized treatment was also influenced by the general health status. The results of this study support the conclusion that periodontal pathogens appear and multiply in the absence of proper hygiene in childhood after the eruption of permanent teeth, and their action leads to the initiation of periodontal diseases.

UNASSIGNED: Tuberculosis (TB) is one of the major global health issues due to its high mortality rate, especially in low- and middle-income countries. One of the key success points of the TB eradication program is early TB diagnosis, which requires rapid and accurate diagnostic testing. This study aimed to evaluate the performance of a newly developed RT-PCR kit (Indigen MTB/DR-TB RT-PCR) in a routine TB clinical setting.
UNASSIGNED: A multi-fluorescence RT-PCR assay was designed and developed to detect regions within IS6110, rpoB, katG, and inhA of the Mycobacterium tuberculosis (MTB) genes. Sputum specimens were obtained from suspected TB patients who visited TB healthcare facilities in two major cities of Indonesia from September 2022 to May 2023. Specimens were assessed using Indigen MTB/DR-TB RT-PCR, acid-fast bacillus (AFB) smear microscopy, MTB culture, and drug susceptibility testing (DST) methods. Fisher\'s exact test (χ2) was used to analyze the Indigen performance relative to culture methods.
UNASSIGNED: The performance of Indigen MTB/DR-TB RT-PCR to detect MTB was assessed using 610 sputum specimens obtained from suspected patients. The overall sensitivity and specificity were 94.12% (95% CI: 90.86-96.48%) and 98.32% (95% CI: 96.20-99.46%), respectively. When the analysis was performed on AFB smear-negative TB subjects (386 subjects), a lower sensitivity level was found at 78.57% (95% CI: 68.26-86.78%), while the specificity level remained similar at 98.34% (95% CI: 96.18-99.46%). The overall performance of Indigen MTB/DR-TB RT-PCR to detect MTB showed substantial agreement with the MTB culture method (kappa value 0.93). In comparison to DST, the sensitivity and specificity levels of Indigen to detect RIF resistance or INH resistance were 78.2% (95% CI: 61.8-90.2%) and 82.8% (95% CI: 64.2-94.2%), respectively, while the specificity level for both groups was at 100% (95% CI, 87.7-100%).
UNASSIGNED: Indigen MTB/DR-TB RT-PCR demonstrated reliable performance for TB molecular diagnostic testing and can be implemented in routine TB diagnostic settings.

The aim of this research was to determine if there are differences in early bacterial adhesion among CAD/CAM dental materials after 24 h exposure in the oral environment. One hundred twenty specimens were prepared according to the manufacturer\'s recommendations and divided into six groups: RBC (resin-based composite), PMMA (polymethyl methacrylate), PEEK (polyether ether ketone), ZP (zirconia polished), ZG (zirconia glazed), and cobalt-chromium alloy (CoCr alloy). Twenty healthy participants were instructed to carry an intraoral device with six specimens, one per group, for 24 h. Thereafter, real-time polymerase chain reaction (qPCR) and scanning electron microscopy (SEM) analyses enabled quantification and 2D view of biofilm formed on the specimens\' surfaces. Kruskal-Wallis test and Dunn\'s post hoc analysis were used for inter-group comparison and data were presented as median (minimum-maximum). RBC specimens accumulated less bacteria, in comparison with ZG (p = 0.017) and PEEK specimens (p = 0.030), that dominated with the highest amount of adhered bacterial biofilm. PMMA, CoCr, and ZP specimens adhered more bacteria than RBC (p > 0.05), and less than ZG (p > 0.05) and PEEK (p > 0.05). The bacterial number varied considerably among participants. The obtained results enable a closer view into the susceptibility of CAD/CAM materials to microorganisms during the presence in the oral environment, which can be beneficial for a proper selection of these materials for a variety of dental restorations.

UNASSIGNED: Salmonella has become one of the hazards prevalent foodborne pathogens causing different diseases in chickens. However, Salmonella typhimurium (ST), a nonhost-specific serovar, is a major avian agent that causes severe disturbance in young chicken wellness.
UNASSIGNED: The occurrence of Salmonella in chickens and their antimicrobial resistance were explored in this study. In addition, the immune response of 1-day-old broiler chicks, against multidrug resistant (MDR) ST infection, was also assessed at 4 and 24 hours post infection (pi) in the cecum and spleen, representing their mucosal and systemic immune responses, respectively.
UNASSIGNED: A total of 375 samples from 130 diseased and apparently healthy broiler and layer chickens were randomly collected for Salmonella isolation, identification, and resistance profile evaluation, from farms and different clinical laboratories. The immune response of 1-day-old broiler chicks, Ross 308, against in-vivo ST infection was ascertained through the evaluation of heterophile phagocytosis and s expression of cytokines, immunoglobulin A and other immune-regulating genes in the cecum and spleen. Twenty-four, 1-day-old nonvaccinated broiler chicks were used and divided into two groups. The chicks in the infected group were orally inoculated with 0.5 ml of 2 × 108 colony forming units (CFU)/ml of MDR ST suspension, while those in the control group were taken nutrient broth.
UNASSIGNED: Seven out of 130 (5.38%) examined chickens were positive for Salmonella. All isolates (100%) were resistant to amoxicillin-clavulanic acid (AMC), cefazolin (CZ), cefoxitin (FOX), ciprofloxacin (CIP), nalidixic acid (NA), tetracycline (TE), fosfomycin (FOS), and colistin (CT) with multiple antimicrobial resistances (MARs) index range of 0.72-0.83, where none of them was resistant to meropenem (MEM). The results of immune response revealed that chicks infected with ST showed significantly different phagocytosis percentages and index values compared to controls. According to the real-time quantitative polymerase chain reaction (RT-qPCR) results, the transcription of IL-8, iNOS, IL-18, IgA, and IFN-γ for chicks infected by ST showed a significantly increased trend (p < 0.01) with increasing chicken age and was higher in the cecum than spleen compared to controls (p < 0.05) during 24 hours after infection.
UNASSIGNED: The findings indicated a strong mucosal immune response in the chicks after the ST challenge, which reflects humoral and cellular responses. Our insight recommended the occurrence of a natural immune response stimulator at 1 day age to face the infection, and this can prevent the resistance transfer, with efficient control measures.

BACKGROUND: Inflammatory myopathy and perivasculitis have been recently described in horses with chronic equine piroplasmosis (EP). These alterations may be linked to poor performances. The aims of this study were to evaluate the prevalence for EP in clinically healthy Italian Standardbred (IS) racehorses and to compare laboratory parameters and performance metrics between positive and negative horses. Real-time PCR was applied for the detection of T. equi and B. caballi positivity. Haematology parameters, blood chemistry results, subjective muscle mass scores, and performance metrics were compared between PCR-positive and -negative horses.
RESULTS: This cross-sectional study included 120 well-trained IS racehorses and was performed over a two-years period. The prevalence of T. equi was 36.3%, whereas all samples were negative for B. caballi. Red blood cells count, haemoglobin concentration, aspartate aminotransferase, alkaline phosphatase, and gamma-glutamyl transferase activities were significantly higher in PCR-positive horses, whereas blood urea nitrogen, globulin concentration and globulin-to-albumin ratio were significantly lower in PCR-positive horses compared to PCR-negative ones. Nonetheless, all values fell within the physiological range. The best racing time, which was selected as the most representative of the performance metrics at the principal component analysis, was not affected by PCR positivity, the muscle mass score or the training yard. The best racing time was significantly better in horses with a mild or no signs of muscular atrophy, within the PCR-positive group. The muscle mass score was associated with the training yard in PCR-negative horses.
CONCLUSIONS: Prevalence of T. equi was high in IS racehorses in southern Italy. The absence of obvious changes in haematological and biochemical parameters, as well as performance metrics in positive horses, highlights the need for specific diagnostic tests to identify chronically infected horses.

UNASSIGNED: Amoebic liver abscess (ALA) is commonly seen in tropical countries and diagnosis of ALA relies mainly on non-specific serological and imaging techniques as well as PCR from pus.
UNASSIGNED: This study evaluated the potential of using cell free DNA (cfDNA) from serum and urine for diagnosing ALA.
UNASSIGNED: We prospectively evaluated quantitative PCR (qPCR) for detection of cf DNA in serum and urine sample in all liver abscess patients. The samples were collected from patients reporting to emergency ward of Postgraduate Institute of Medical Education and Research, Chandigarh, India with symptoms suggestive of liver abscess. Real time PCR was done to detect cf DNA in serum and urine by targeting 99-bp unit of small subunit rRNA of Entamoeba histolytica and conventional PCR for pus.
UNASSIGNED: A total 113 samples (serum and urine) and 100 pus samples were analysed. A total of 62 ALA patients were confirmed; with maximum 57 patients detected by qPCR for cfDNA in the serum, 55 patients by PCR on pus aspirate and 50 ALA patients by qPCR for cfDNA in urine sample. Therefore, the sensitivity of qPCR for detection of cf DNA in serum was 91.94% and for urine was 80.65%.
UNASSIGNED: A total of 11.2% of ALA patients were diagnosed only through detection of E. histolytica cf DNA in their serum and urine. Detection of cfDNA from serum, urine of ALA has a potential role in future especially for developing countries as it is a rapid, sensitive and patient friendly diagnostic approach.

OBJECTIVE: Longer-term intake of fatty acid (FA)-modified dairy products (SFA-reduced, MUFA-enriched) was reported to attenuate postprandial endothelial function in humans, relative to conventional (control) dairy. Thus, we performed an in vitro study in human aortic endothelial cells (HAEC) to investigate mechanisms underlying the effects observed in vivo.
METHODS: This sub-study was conducted within the framework of the RESET study, a 12-week randomised controlled crossover trial with FA-modified and control dairy diets. HAEC were incubated for 24 h with post-intervention plasma samples from eleven adults (age: 57.5 ± 6.0 years; BMI: 25.7 ± 2.7 kg/m2) at moderate cardiovascular disease risk following representative sequential mixed meals. Markers of endothelial function and lipid regulation were assessed.
RESULTS: Relative to control, HAEC incubation with plasma following the FA-modified treatment increased postprandial NOx production (P-interaction = 0.019), yet up-regulated relative E-selectin mRNA gene expression (P-interaction = 0.011). There was no impact on other genes measured.
CONCLUSIONS: Incubation of HAEC with human plasma collected after longer-term dairy fat manipulation had a beneficial impact on postprandial NOx production. Further ex vivo research is needed to understand the impact of partial replacement of SFA with unsaturated fatty acids in dairy foods on pathways involved in endothelial function.

Giardia duodenalis is a common pathogenic intestinal protozoan parasite with high prevalence in developing countries, especially among children. The distribution of giardia assemblages among humans and their clinical relevance remains controversial. This study aimed to determine the prevalence and assemblage of Giardia among children under 5 years of age in Jimma, Southwest Ethiopia. Employing a case-control design, 606 children presenting with diarrhea at Jimma university medical center and Serbo Health Center were enrolled from December 2016 to July 2018 along with 617 matched controls without diarrhea. Giardia was detected and typed using real-time PCR. Univariate and multivariate regression analysis was performed. The total prevalence of Giardia was 41% (501/1223) and did not differ significantly between cases and controls (40% vs 42%). Prevalence increased by age, with the highest prevalence seen in children aged ≥ 25 months. Children without diarrhea with a history of diarrhea during the last month were more likely to be Giardia positive compared to children with no history diarrhea (OR 1.8 and 95%CI; 1.1-2.9). Regardless of current diarrhea symptoms, assemblage B predominated with 89%, followed by assemblage A (8%) and mixed infection assemblage A and B (3%). We report a high prevalence of Giardia by PCR detection in Jimma, Ethiopia, with assemblage B being predominant. There was a similar distribution of Giardia assemblages between children with and without diarrhea. Increasing age was a risk factor for Giardia infection. Community-based prevention and control strategies need to be employed to decrease the risk of giardia infection.